Biomedical applications of colloidal solutions of magnetic particles, or ferrofluids, have developed essentially in three directions:
imaging (MRI) as contrast agents (1, 2, 3),
magnetic separation of various cells, organites or biological molecules (4, 5, 6, 7, 8, 9)
destruction of target cells by creation of a local hyperthermia under pulsating magnetic field (10).
The most extensively employed magnetic particles are the ferrites MFe.sub.2 O.sub.4 (including magnetite Fe.sub.3 O.sub.4) and maghemite .gamma. Fe.sub.2 O.sub.3. The surface of the particles must be conditioned in order to obtain colloidal solutions that are stable in physiological medium. In most cases the particles are coated with macromolecules such as carbohydrates like dextran (10, 12, 1), proteins like albumin (5, 8) or synthetic polymers like methacrylates and organosilanes (7, 9, 13). However, as Groman indicates, covering the particles with macromolecules of high molecular mass does not make it possible to obtain sols that are stable in the long term. The macromolecules separate from the particles, which then gradually aggregate. Other methods of conditioning the surface of the particles have been proposed, such as the use of molecules of low molecular mass, containing complexing groups such as phosphates, phosponates and carboxylates (2). Very particularly, hydroxylated polycarboxylic acids such as citric and tartaric acids and polycarboxylic acids containing thiol groups, like dimercaptosuccinic acid (DMSA) are complexed with the surface atoms of iron(III) (14). Each of these complexing molecules is bonded to one or more surface sites of the particles. The aqueous sols thus obtained are very stable in physiological conditions.
In many biomedical applications of the magnetic particles the latter must be coupled to a specific protein. Thus, in the case of cell sorting under magnetic field the particles must be capable of bonding specifically to the target cells. This recognition is often ensured by the formation of an antigen-antibody complex between a surface antigen of the target cell and an antibody bonded to the particles. The protein may be either adsorbed directly at the surface of the particles (4) or bonded covalently (5, 7, 13). The use of difunctional intermediate compounds such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (8, 14) makes it possible to bond an antibody strongly to a particle via a peptide bond and a disulfide bridge, without damage to the protein or to its antigenic properties (15).
French patent No. 2 662 539 (14) described a process for obtaining finely divided magnetic supports by controlled modification of particles filled with precursor ferrofluid, including a stage of treatment with an agent capable of modifying the nature of the surface of the said particles, and so as to make them stable in polar or nonpolar solvents in wide pH ranges and to endow them with a chemical or biological reactivity. An example of such a modification is a DMSA-based ferrofluid making it possible to render thiol groups available and reactive at the surface of these so-called substituted ferrofluids; however, in the usual pH and ionic strength conditions, these thiol groups tend to become oxidized and to form disulfide bridges making these ferrofluids ill-suited to a use as constituents of a magnetic effector.
A use, especially for differentiating and/or separating cells which have at their surface a receptor for a ligand signaling a property or a particular physiological state, made it necessary to improve the ferrofluids of patent 2 662 539 (14) in such a way that disulfide bridges do not limit the coupling; Such an improvement then making it possible to produce DMSA ferrofluid complexes coupled covalently to the said effectors and then to employ them as means for differentiating and separating complexes or cells carrying, if appropriate, the receptor for the said effector.
The effector/receptor affinity pairs are numerous and the invention as described below will be capable of being easily applied by a person skilled in the art to the pairs of receptors of interest to him/her, as well as to other affinity pairs such as antibody/antigen, lectin/polysaccharide, biotin/avidin and nucleic acid (+ and - strands) pairs and the like, as soon as the effector can be coupled to the particles through the intermediacy of a difunctional reactant optionally comprising thiol residues one of the functional groups of which permits the bonding to the effector and the other functional group of which is capable of forming S--S, C--S, C--C or C--N bonds with DMSA.
A particular application of the use of the ferrofluid coupled to an effector is the use as effector of an annexine, like annexine V, which has a particular affinity, in the presence of calcium ions, for anionic phospholipids, like phosphatidylserine (PS) and in, a lesser degree, phosphatidylethanolamine (PE). These compounds of the plasmic membranes are essentially localized in the inner layer of the membranes, in contrast to other phosphatidylcholine and sphingo-myelin type compounds which are, on the contrary, predominantly localized in the outer layer of the plasmic membranes. The cell membranes exhibit an asymmetry such that, when the membrane is healthy, the phosphatidylserines and some of the phosphatidyl-ethanolemines residing on the inner layer are inaccessible, whereas, when the membranes are perturbed, there is a random localization of these same phospholipids. Consequently, those that are localized in the inner layer of the membranes become accessible over the whole cells and, in particular, phosphatidyl-serine becomes accessible to a coupling by an annexine like annexine V or capable of being attacked by an external phospholipase. This perturbation may be due to an inflammation, to an apoptosis (16), to autoimmune reactions (17), to a sickle cell anemia (18, 19), or to a pathological or infectious state of the individual (19, 22), or simply to an excessive aging of the cells and especially of the blood cells during their storage in vitro (23). It is thought that the presence of phosphatidylserine in the outer layer of the plasmatic membrane constitutes a signal for the elimination of the cell by the immune system (24, 25).
Patent application WO 91/09628 relates to the use of anticoagulant polypeptides of the annexine class which are provided with a marker and these marked annexines or VAC (vascular anticoagulant protein) are employed as means for differentiating phosphatidyl-serines from phosphatidylcholines and for thus diagnosing a prethrombotic state via the presence of a coupling of annexine to the cells exhibiting a phosphatidylserine in an accessible manner.
Annexines are a group of homologous proteins, from 35 to 45 k daltons, which are found in all mammals at different stages of development, as well as in other vertebrates such as arthropods, slime molds (26), yeasts, sponges, fungi, protozoa, plants and bacteria (27, 28). All mammalian cells, with the exception of erythrocytes, produce an annexine like annexine V. The structure of annexines and their properties are described in reviews (28, 29). These proteins have been sequenced and some of them are available as recombinant proteins (27). As set out above, cell membranes which have been damaged by a mechanism of some kind exhibit an increased capacity for binding annexines; this can be observed, for example, by comparing fresh erythrocytes with erythrocytes which have been subjected to a relatively long storage period. Insofar as this increase in capacity for bonding to annexine is the reflection of an anomaly, the measurement of the erythrocytes' capacity for coupling to annexine is found to be an important tool for the control of quality of the blood to be employed in blood transfusions.
Selection of blood for transfusion is done essentially using three criteria: the immunological compatibility of the donor and of the receiver, the absence of viral or parasitic contamination, and the absence of pathology in the donor. Suitable tests have been developed for verifying blood groups and possible viral or parasitic contaminations; the donor's state of health is evaluated simply by interrogation. It is of interest, however, to note that there is nothing in existence for controlling the degradation of the blood during its preservation, it being known that individual bloods can age at different rates. Another potential problem which can arise in the selection of the blood for transfusion is that of its control when a number of viruses or of pathogenic agents are still unknown and hence undetectable.